Fig. 2: Specificity of dynamic FRET-FISH.

a Sequences of let-7a point mutants. Red letters indicate mutation sites. b Single-molecule images of wild type let-7a and its point mutants. To generate the images, the acceptor signal was accumulated for 60 s at Alexa-488 excitation (blue) and Cy3 excitation (green). Spots where blue and green colors overlap were colored in white. c Representative acceptor fluorescence intensity time traces of let-7a point mutants. d Sequences of let-7 family miRNAs. Red letters indicate sequence variations from let-7a. e False positive rates of let-7 family miRNA detection. For the experiments, we immobilized 100 pM target miRNAs and injected the designated probe sets. Molecules were identified as a proper target miRNA if an acceptor (Cy5) fluorescence signal occurs during the initial 10 s at both Alexa-488 and Cy3 excitations. The false positive rates were calculated by dividing the off-target number by the on-target number. The Numbers in parentheses are the total molecules that we analysed in the experiments.